Objective:To study the protective effect of paeonol on LPS-induced inflammatory injury of macrophage RAW264.7 cells and its mechanism.Methods:Macrophage RAW264.7 cells were cultured and divided into blank group,LPS(1...Objective:To study the protective effect of paeonol on LPS-induced inflammatory injury of macrophage RAW264.7 cells and its mechanism.Methods:Macrophage RAW264.7 cells were cultured and divided into blank group,LPS(1μg/mL)group,paeonol(240μmol/mL)group and TAK242(10μmol/mL)group.The cell activity was detected by CCK8 method,the cell morphology was observed by inverted microscope,the contents of GSH and MDA in cell culture medium were determined by colorimetry,the mitochondrial membrane potential was detected by JC-1 method,the expression distribution of F4/80 and p-NF-κB protein was detected by immunofluorescence method,and the expression of TLR4/MAPK/NF-κB related pathway protein was detected by Western blotting.Results:Compared with the blank group,the cell viability induced by 1μg/mL LPS was 0.4972±0.061(P<0.01),which was close to the half inhibition rate.Compared with LPS group,the expression of p-NF-κB protein in 240μmol/mL paeonol pretreated cell group was down-regulated most significantly(P<0.01),and the expression of TLR4 protein was inhibited most significantly in 10μmol/mL TAK242 pretreated cell group.Compared with LPS group(P<0.01),the cell morphology of paeonol group recovered.Decrease MDA content and increase GSH content in cell culture medium(P<0.01),In the results of mitochondrial membrane potential,the red light of paeonol group was significantly enhanced and the green light was significantly weakened(P<0.001).The expression distribution of F4/80 and p-NF-κB protein in paeonol group decreased significantly(P<0.01),and the expressions of TLR4,p-IκB,p-p38,p-JNK and p-NF-κB protein were down-regulated(P<0.05).Conclusion:Paeonol can improve the inflammatory injury of RAW264.7 cells induced by LPS,and its mechanism may be related to TLR4/MAPK/NF-κB pathway.展开更多
Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide ...Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.展开更多
背景:前期研究表明葛根素干预后破骨细胞的分化被抑制,Notch1、HES1、Jagged1等Notch信号通路相关蛋白表达量下降,但Notch1信号通路对于葛根素抑制破骨细胞分化的作用机制尚不明确。目的:探究Notch信号通路对葛根素抑制小鼠巨噬细胞Raw2...背景:前期研究表明葛根素干预后破骨细胞的分化被抑制,Notch1、HES1、Jagged1等Notch信号通路相关蛋白表达量下降,但Notch1信号通路对于葛根素抑制破骨细胞分化的作用机制尚不明确。目的:探究Notch信号通路对葛根素抑制小鼠巨噬细胞Raw264.7分化为破骨细胞的影响。方法:将Raw264.7细胞分为7组干预培养,空白对照组采用DMEM高糖完全培养基培养,破骨细胞诱导组采用破骨诱导培养基培养,葛根素干预组在破骨诱导的同时加入50μmol/L葛根素培养,葛根素+Notch1 si RNA对照组、葛根素+Notch1 si RNA组、葛根素+Notch1过表达对照组、葛根素+Notch1过表达组分别采用Notch1 si RNA对照序列、Notch1 si RNA序列、Notch1过表达对照质粒、Notch1过表达质粒转染Raw264.7细胞后,加入破骨诱导培养基和葛根素进行培养。培养7 d后,采用抗酒石酸酸性磷酸酶染色观察破骨细胞的数量和大小,F-actin染色观察破骨细胞骨架形成情况,RT-PCR检测破骨细胞形成标志物的基因表达水平。结果与结论:(1)抗酒石酸酸性磷酸酶染色显示:葛根素干预可以抑制破骨细胞的生成,Notch1沉默会进一步减少破骨细胞的生成数量,Notch1过表达后破骨细胞生成数量明显增加;(2)F-actin染色显示:Raw264.7细胞经破骨诱导可以形成边界清晰的F-actin环,葛根素干预会抑制细胞骨架的形成,Notch1沉默会增强葛根素的抑制作用,而Notch1过表达则能减弱葛根素的抑制作用;(3)RT-PCR检测显示,葛根素可以抑制抗酒石酸酸性磷酸酶、组织蛋白酶K和c-Fos的m RNA表达,Notch1沉默后上述3个因子的m RNA表达进一步降低,Notch1过表达后上述3个因子的m RNA表达增加。结果表明:Notch信号通路在Raw264.7细胞分化为破骨细胞的过程中发挥作用,葛根素通过抑制Notch信号通路抑制Raw264.7细胞分化为破骨细胞。展开更多
背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供...背景:溶质载体家族1成员5(solute carrier family 1 member 5,SLC1A5)在多种疾病中发挥了潜在作用,但确切作用机制尚不清楚。构建稳定的SLC1A5过表达和敲低细胞模型可为深入研究SLC1A5在疾病中的确切作用机制以及发现潜在治疗靶点提供有力的实验工具。目的:构建小鼠SLC1A5过表达和敲低的慢病毒载体,以建立稳定转染的RAW264.7细胞株,为深入探讨SLC1A5在炎症中的作用提供实验基础。方法:根据SLC1A5基因序列设计合成引物并使用聚合酶链反应扩增该基因片段。将目的基因定向接入经Age I/Nhe I酶切的载体质粒GV492中构建重组慢病毒质粒,对阳性克隆进一步筛选后测序比对结果;pHelper1.0质粒载体、pHelper2.0质粒载体、目的质粒载体与293T细胞共同培养并转染,获得慢病毒原液进行包装和滴度测定;在此基础上,通过体外培养RAW264.7细胞,确定嘌呤霉素工作质量浓度;不同滴度的慢病毒分别与RAW264.7细胞共同培养,根据荧光强度确定转染效率;用嘌呤霉素挑选出稳定转染细胞,实时荧光定量聚合酶链反应和蛋白免疫印迹方法检测稳定转染细胞株的SLC1A5基因和蛋白表达水平。结果与结论:(1)测序序列与目的序列一致提示重组慢病毒载体构建成功;(2)过表达SLC1A5慢病毒的滴度为1×10~9 TU/mL,敲低SLC1A5慢病毒的滴度为3×10~9 TU/mL;(3)确定RAW264.7细胞嘌呤霉素工作质量浓度为3μg/mL;(4)过表达/敲低SLC1A5慢病毒转染RAW264.7细胞的最佳条件皆为HiTransG P转染增强液且感染复数值等于50;(5)过表达SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量明显上调,而敲低SLC1A5稳转细胞株中SLC1A5基因和蛋白的表达量显著下调。结果表明,成功构建了小鼠SLC1A5过表达和敲低的慢病毒载体并获得稳定转染的RAW264.7细胞株。展开更多
Objective: The effect of Chuanzhi Fang (ZGC) on the whole genome expression profile of RAW264.7 cells activated by lipopolysaccharide (LPS) was analyzed, and to explore the possible mechanism of action and core target...Objective: The effect of Chuanzhi Fang (ZGC) on the whole genome expression profile of RAW264.7 cells activated by lipopolysaccharide (LPS) was analyzed, and to explore the possible mechanism of action and core target of this formula on macrophage inflammatory injury at the overall level. Methods: A model of LPS-induced inflammation in RAW264.7 cells was constructed, and the effect of ZGC intervention on the genome-wide expression of inflammatory macrophages 3was examined by gene microarray technology, GO/KEGG enrichment analysis was performed for significantly differentially expressed genes among each group. Results: The results of genome-wide expression profiling microarray analysis showed that the ZGC intervention group upregulated the expression of 5 genes including C4bp and inhibited the expression of 22 genes including Mgat3, Psma6, and Siglecg relative to the LPS model group. KEGG signaling pathway analysis results showed that ZGC mainly acted through cytokine receptor interaction and the C-type lectin receptor signaling pathway. Conclusion: ZGC can interfere with the abnormal expression of 27 genes in inflammatory macrophages, and the related genes may exert corresponding anti-inflammatory effects by affecting cytokine receptor interactions, C-type lectin receptor signaling pathway, and TLR4/ NF-κB signaling pathway.展开更多
文摘Objective:To study the protective effect of paeonol on LPS-induced inflammatory injury of macrophage RAW264.7 cells and its mechanism.Methods:Macrophage RAW264.7 cells were cultured and divided into blank group,LPS(1μg/mL)group,paeonol(240μmol/mL)group and TAK242(10μmol/mL)group.The cell activity was detected by CCK8 method,the cell morphology was observed by inverted microscope,the contents of GSH and MDA in cell culture medium were determined by colorimetry,the mitochondrial membrane potential was detected by JC-1 method,the expression distribution of F4/80 and p-NF-κB protein was detected by immunofluorescence method,and the expression of TLR4/MAPK/NF-κB related pathway protein was detected by Western blotting.Results:Compared with the blank group,the cell viability induced by 1μg/mL LPS was 0.4972±0.061(P<0.01),which was close to the half inhibition rate.Compared with LPS group,the expression of p-NF-κB protein in 240μmol/mL paeonol pretreated cell group was down-regulated most significantly(P<0.01),and the expression of TLR4 protein was inhibited most significantly in 10μmol/mL TAK242 pretreated cell group.Compared with LPS group(P<0.01),the cell morphology of paeonol group recovered.Decrease MDA content and increase GSH content in cell culture medium(P<0.01),In the results of mitochondrial membrane potential,the red light of paeonol group was significantly enhanced and the green light was significantly weakened(P<0.001).The expression distribution of F4/80 and p-NF-κB protein in paeonol group decreased significantly(P<0.01),and the expressions of TLR4,p-IκB,p-p38,p-JNK and p-NF-κB protein were down-regulated(P<0.05).Conclusion:Paeonol can improve the inflammatory injury of RAW264.7 cells induced by LPS,and its mechanism may be related to TLR4/MAPK/NF-κB pathway.
基金supported by the National Natural Science Foundation of China,No.32371048(to YK)the Peking University People’s Hospital Research and Development Funds,No.RDX2021-01(to YK)the Natural Science Foundation of Beijing,No.7222198(to NH)。
文摘Macrophages play an important role in peripheral nerve regeneration,but the specific mechanism of regeneration is still unclear.Our preliminary findings indicated that neutrophil peptide 1 is an innate immune peptide closely involved in peripheral nerve regeneration.However,the mechanism by which neutrophil peptide 1 enhances nerve regeneration remains unclear.This study was designed to investigate the relationship between neutrophil peptide 1 and macrophages in vivo and in vitro in peripheral nerve crush injury.The functions of RAW 264.7 cells we re elucidated by Cell Counting Kit-8 assay,flow cytometry,migration assays,phagocytosis assays,immunohistochemistry and enzyme-linked immunosorbent assay.Axonal debris phagocytosis was observed using the CUBIC(Clear,Unobstructed Brain/Body Imaging Cocktails and Computational analysis)optical clearing technique during Wallerian degeneration.Macrophage inflammatory factor expression in different polarization states was detected using a protein chip.The results showed that neutrophil peptide 1 promoted the prolife ration,migration and phagocytosis of macrophages,and CD206 expression on the surfa ce of macrophages,indicating M2 polarization.The axonal debris clearance rate during Wallerian degeneration was enhanced after neutrophil peptide 1 intervention.Neutrophil peptide 1 also downregulated inflammatory factors interleukin-1α,-6,-12,and tumor necrosis factor-αin invo and in vitro.Thus,the results suggest that neutrophil peptide 1 activates macrophages and accelerates Wallerian degeneration,which may be one mechanism by which neutrophil peptide 1 enhances peripheral nerve regeneration.
文摘背景:前期研究表明葛根素干预后破骨细胞的分化被抑制,Notch1、HES1、Jagged1等Notch信号通路相关蛋白表达量下降,但Notch1信号通路对于葛根素抑制破骨细胞分化的作用机制尚不明确。目的:探究Notch信号通路对葛根素抑制小鼠巨噬细胞Raw264.7分化为破骨细胞的影响。方法:将Raw264.7细胞分为7组干预培养,空白对照组采用DMEM高糖完全培养基培养,破骨细胞诱导组采用破骨诱导培养基培养,葛根素干预组在破骨诱导的同时加入50μmol/L葛根素培养,葛根素+Notch1 si RNA对照组、葛根素+Notch1 si RNA组、葛根素+Notch1过表达对照组、葛根素+Notch1过表达组分别采用Notch1 si RNA对照序列、Notch1 si RNA序列、Notch1过表达对照质粒、Notch1过表达质粒转染Raw264.7细胞后,加入破骨诱导培养基和葛根素进行培养。培养7 d后,采用抗酒石酸酸性磷酸酶染色观察破骨细胞的数量和大小,F-actin染色观察破骨细胞骨架形成情况,RT-PCR检测破骨细胞形成标志物的基因表达水平。结果与结论:(1)抗酒石酸酸性磷酸酶染色显示:葛根素干预可以抑制破骨细胞的生成,Notch1沉默会进一步减少破骨细胞的生成数量,Notch1过表达后破骨细胞生成数量明显增加;(2)F-actin染色显示:Raw264.7细胞经破骨诱导可以形成边界清晰的F-actin环,葛根素干预会抑制细胞骨架的形成,Notch1沉默会增强葛根素的抑制作用,而Notch1过表达则能减弱葛根素的抑制作用;(3)RT-PCR检测显示,葛根素可以抑制抗酒石酸酸性磷酸酶、组织蛋白酶K和c-Fos的m RNA表达,Notch1沉默后上述3个因子的m RNA表达进一步降低,Notch1过表达后上述3个因子的m RNA表达增加。结果表明:Notch信号通路在Raw264.7细胞分化为破骨细胞的过程中发挥作用,葛根素通过抑制Notch信号通路抑制Raw264.7细胞分化为破骨细胞。
基金Chinese Academy of Traditional Chinese Medicine Autonomous Topic Selection Project(No.ZZ2018017)Research Development Fund Project of the Medical Experimental Center of the Chinese Academy of Traditional Chinese Medicine(No.FZ2023003)。
文摘Objective: The effect of Chuanzhi Fang (ZGC) on the whole genome expression profile of RAW264.7 cells activated by lipopolysaccharide (LPS) was analyzed, and to explore the possible mechanism of action and core target of this formula on macrophage inflammatory injury at the overall level. Methods: A model of LPS-induced inflammation in RAW264.7 cells was constructed, and the effect of ZGC intervention on the genome-wide expression of inflammatory macrophages 3was examined by gene microarray technology, GO/KEGG enrichment analysis was performed for significantly differentially expressed genes among each group. Results: The results of genome-wide expression profiling microarray analysis showed that the ZGC intervention group upregulated the expression of 5 genes including C4bp and inhibited the expression of 22 genes including Mgat3, Psma6, and Siglecg relative to the LPS model group. KEGG signaling pathway analysis results showed that ZGC mainly acted through cytokine receptor interaction and the C-type lectin receptor signaling pathway. Conclusion: ZGC can interfere with the abnormal expression of 27 genes in inflammatory macrophages, and the related genes may exert corresponding anti-inflammatory effects by affecting cytokine receptor interactions, C-type lectin receptor signaling pathway, and TLR4/ NF-κB signaling pathway.